title: Asparagine repeat function in a Plasmodium falciparum protein assessed via a regulatable fluorescent affinity tag.
abstract: One in four proteins in Plasmodium falciparum contains asparagine repeats. We probed the function of one such 28-residue asparagine repeat present in the P. falciparum proteasome lid subunit 6, Rpn6. To aid our efforts, we developed a regulatable, fluorescent affinity (RFA) tag that allows cellular localization, manipulation of cellular levels, and affinity isolation of a chosen protein in P. falciparum. The tag comprises a degradation domain derived from Escherichia coli dihydrofolate reductase together with GFP. The expression of RFA-tagged proteins is regulated by the simple folate analog trimethoprim (TMP). Parasite lines were generated in which full-length Rpn6 and an asparagine repeat-deletion mutant of Rpn6 were fused to the RFA tag. The knockdown of Rpn6 upon removal of TMP revealed that this protein is essential for ubiquitinated protein degradation and for parasite survival, but the asparagine repeat is dispensable for protein expression, stability, and function. The data point to a genomic mechanism for repeat perpetuation rather than a positive cellular role. The RFA tag should facilitate study of the role of essential genes in parasite biology.
authors.affiliation: Departments of Medicine and Molecular Microbiology, Howard Hughes Medical Institute, Washington University School of Medicine, St Louis, MO 63110, USA.
authors.forename: Vasant
authors.initials: V
authors.lastname: Muralidharan
authors.forename: Anna
authors.initials: A
authors.lastname: Oksman
authors.forename: Mari
authors.initials: M
authors.lastname: Iwamoto
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Wandless
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Goldberg
mesh_terms: D001216:Asparagine / Q000378:metabolism*; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects / Q000469:parasitology; D005456:Fluorescent Dyes / Q000378:metabolism*; D020043:Genes, Essential / Q000235:genetics; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010963:Plasmodium falciparum / Q000187:drug effects / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D055550:Protein Stability / Q000187:drug effects; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D015203:Reproducibility of Results; D017384:Sequence Deletion / Q000235:genetics; D014295:Trimethoprim / Q000494:pharmacology; D054875:Ubiquitination / Q000187:drug effects
chemical_list: D005456:Fluorescent Dyes; D050505:Mutant Proteins; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D001216:Asparagine; D014295:Trimethoprim; D046988:Proteasome Endopeptidase Complex